Protein kinase C (PKC) isozymes have been implicated in numerous cellular signaling pathways and alterations in their activities contribute to many disease processes, including carcinogenesis and altered pain sensitivity. Drugs targeting individual isozymes have significant potential for treatment of some of these disease states; however, isozyme-specific drugs largely are lacking. A 3D crystal structure of PKC also is lacking and, once obtained, will reveal the enzyme in its closed, inactive form. In the past grant period, we have grown 2D crystals of several isozymes on an activating lipid monolayer and resolved some structural differences in PKCdelta+/- substrate. Recent work has improved the resolution to 5-6 A by electron microscopy, 2-3 A by electron diffraction. Preliminary analysis of a tilt series of 2D crystals has permitted preliminary generation of a 3D reconstruction. We also characterized biphasic activation/inhibition of PKC by lipids and anesthetics. We propose to refine the structures of PKCdelta and PKCbeta in the presence and absence of various activators and inhibitors. Our hypothesis is that changes in PKC activity that accompany interaction with substrates, activators, and inhibitors are reflected in changes in conformation and lattice packing that can be observed in 2D crystals on lipid monolayers. Understanding these changes will contribute to the design of better isozyme-specific activators and inhibitors. Our specific aims are: I) To generate a high-resolution ion 3Dmodel of PKCdelta from electron micrographs of 2D crystals analyzed at various tilt angles. II) To characterize changes in PKC8 in response to different PKC- and lipid-binding molecules including: A) different active site substrates and inhibitors; B) molecules binding other PKCdelta domains; and C) different lipid-soluble (e.g. alcohols) or lipid binding (e.g. local anesthetics) molecules. III) To extend the structural analysis to a calcium-dependent isozyme, PKCbeta, and to examine effects of calcium and C2-binding molecules on the structure. If time permits, examination of additional isozymes will be initiated.